Genetic Barcodes for Improved Environmental Tracking of an Anthrax Simulant 1

Patricia Buckley1, Bryan Rivers1,2, Sarah Katoski1,2, Michael H. Kim1, F. Joseph Kragl1, Stacey 2

Broomall1, Michael Krepps1,3, Evan W. Skowronski1% , C. Nicole Rosenzweig1, Sari Paikoff4, 3

Peter Emanuel1, Henry S. Gibbons1# 4

1Biosciences Division, US Army Edgewood Chemical Biological Center, Aberdeen Proving 5

Ground, MD, USA 6

2Science Applications International Corporation, Aberdeen Proving Ground, MD, USA 7

3Excet, Inc., Aberdeen Proving Ground, MD, USA 8

4Defense Threat Reduction Agency, Ft. Belvoir, VA, USA 9

%Current address: TMG Biosciences, Incline Village, NV, USA. 10

Running title: Chromosomal Barcoding of Anthrax Simulants 11

Corresponding Author: henry.s.gibbons.civ@mail.mil 12

13

Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.01827-12 AEM Accepts, published online ahead of print on 21 September 2012

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Abstract 14

The development of realistic risk models that predict the dissemination, dispersion and 15

persistence of potential biothreat agents have utilized non-pathogenic surrogate organisms such 16

as Bacillus atrophaeus var. globigii (“BG”) or commercial products such as B. thuringiensis 17

serovar kurstaki (Btk). Comparison of results from outdoor tests under different conditions 18

requires the use of genetically identical strains; however the requirement for isogenic strains 19

limits the ability to compare other desirable properties, such as the behavior in the environment 20

of same strain prepared using different methods. Finally, current methods do not allow long-21

term studies of persistence or reaerosolization in test sites where simulants are heavily used or in 22

areas where Btk is applied as a biopesticide. To create a set of genetically heterogeneous yet 23

phenotypically indistinguishable strains so that variables intrinsic to simulations (e.g. sample 24

preparation) can be varied and the strains can be tested under otherwise identical conditions, we 25

have developed a strategy to introduce small genetic signatures (“barcodes”) into neutral regions 26

of the genome. The barcodes are stable over 300 generations and do not impact in vitro growth 27

or sporulation. Each barcode contains common and specific Tags that allow differentiation of 28

marked strains from wild-type strains and from each other. Each Tag is paired with specific real-29

time PCR assays that facilitate discrimination of barcoded strains from wild-type strains and 30

from each other. These uniquely barcoded strains will be valuable tools for research into the 31

environmental fate of released organisms by providing specific artificial detection signatures. 32

Introduction 33

Spores of Bacillus anthracis (BA), the causative agent of anthrax disease, have been successfully 34

weaponized on large scales in at least two historical offensive biological weapons programs (1, 35

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18, 40, 47); were disseminated through the mail in the well-documented 2001 anthrax attacks (6, 36

25-26, 38); and were alleged to have been used as a weapon in the former Rhodesia (29, 32). For 37

this reason, BA remains classified as a Category A biothreat agent. Their physical hardiness, 38

resistance to heat and environmental insults, and the relative ease with which spores could be 39

refined, milled, and aerosolized without significant loss of viability made BA highly desirable as 40

a weapon. Its historical use as a weapon or bioterrorism agent and the substantial potential 41

economic consequences of anthrax releases have made understanding the behavior and dynamics 42

of Bacillus spores a major focus of research, as knowledge of spore persistence, dissemination, 43

and behavior under decontamination regimens is critical to developing accurate risk models and 44

response regimens that are sufficiently robust while minimizing social and economic disruption. 45

Despite the clear need to acquire knowledge about BA itself, its virulent nature by multiple routes 46

of infection make use of the actual agent (or even attenuated derivatives) in outdoor tests 47

impossible. For this reason, initial efforts to develop non-pathogenic bacterial species as 48

simulants focused on B. atrophaeus var. globigii (BG), a relative of B. subtilis (13, 19). BG has 49

been used over many years as an outdoor simulant of BA (34). However, subsequent research 50

has shown that, while BG does mimic many of the properties of B. anthracis, it lacks an 51

exosporium and has different thermal-kill properties (8, 12) that decrease its utility as a simulant 52

for BA. The repertoire of BG strains in use is quite small, and is restricted to a single lineage 53

with very few available polymorphisms that can discriminate strains, many of which may affect 54

strain and/or spore phenotypes (13). 55

The limitations of BG as a surrogate for BA have prompted several groups to evaluate B. 56

thuringiensis (Bt) serovars as potential anthrax surrogates (12, 17). Like BG, Bt strains are not 57

known to cause disease in humans, and many strains are available off-the-shelf as biological 58

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pesticides for widespread agricultural use in conventional and organic insect pest control (10). 59

Following widespread outdoor applications in pest-control scenarios, BT serovar kurstaki (Btk) 60

strains have been recovered from asymptomatic individuals following widespread aerial spray 61

applications over populated areas (44-45) without any concurrent epidemiological signs of Btk-62

associated disease (11)While Bt and its virulent phylogenetic neighbors B. cereus and B. 63

anthracis share a highly conserved core genome, the accessory or pan-genome is quite variable 64

(28, 36) and consists mainly of phages and plasmids, which encode most of the strain-specific 65

functions that dictate host tropism (e.g. capsule, toxins). The crystalline toxins expressed by BT 66

strains are specific to insects and are not known to affect mammalian hosts. Thus, BT spores 67

share many of the important physical and biochemical characteristics of anthrax spores but do 68

not pose a biological hazard to humans. While the use of B. thuringiensis as an anthrax simulant 69

is not a novel idea (United Nations inspectors recovered a toxin-less strain from a suspect 70

bioweapons facility in Iraq in the late 1990’s (9)), it has not yet been widely adopted. The 71

widespread application of BT (particularly serovar kurstaki, or Btk) as a biopesticide has recently 72

facilitated experimental studies of the persistence and transport of BT in the environment (45-73

46). While those studies have provided extremely valuable information about the life-cycle of 74

deliberately released BT spores, the agricultural application of commercial BT preparations may 75

not mimic the anticipated aerosol dissemination of an authentic BW agent, confounding the 76

ability to develop realistic models. 77

Gathering accurate information about organism behavior in the environment requires a 78

combination of robust and reproducible sampling techniques, rigorous methodology, and 79

optimally, a well-characterized input strain. Until now, only very limited numbers of suitable 80

strains existed, limiting the number of possible studies in any given area or time until the 81

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recoverable signature returned to background levels. Particularly with persistent spores and in 82

heavily used areas such as the US Army’s Dugway Proving Ground, low-level positive signals 83

could be either authentic or spurious, resulting from potential reaerosolization of spores left over 84

from previous tests. In fact, the level of residual BG spores in the soils at Dugway Proving 85

Ground is as high as 105 spores/g soil (K. Omberg, personal communication). The lack of 86

specific signatures for any given strain has made the differentiation of those events impossible. 87

As a potential solution to this problem, we describe here a new approach to simulant 88

development whereby a stable genetic tag, or “Barcode,” is integrated directly into the 89

chromosome of a Btk strain. Each Barcode contains two Tag modules, one common to all 90

barcoded strains and one specific for each strain. To facilitate the detection and quantitation of 91

each barcoded strain, tag-specific real-time PCR assays are presented that can distinguish the 92

strains from each other, from wild-type strains, and from a panel of near-neighbors and other 93

potential interferents. We present data on the stability of the barcode during serial transfer and 94

show that the insertion is neutral toward in vitro growth kinetics. The development of new, 95

specific strains will have dramatic impacts on the methodology of testing and analysis of 96

environmental releases. 97

Materials and methods 98

Strains and plasmids – Strains and plasmids utilized in this study are shown in Table 1. Since 99

the tagged spore was foreseen to be used in a broad range of indoor and outdoor test scenarios, 100

strain ATCC 33679, an HD-1 strain (serotype 3a3b) that is registered with the United States 101

Environmental Protection Agency as an approved biopesticide, was selected as the backbone for 102

the barcoding efforts for its outstanding safety record in widespread Gypsy Moth control efforts, 103

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with annual outdoor applications of ~453 metric tonnes of Btk spores applied over >138,000 Ha 104

in the United States alone with no significant medical issues recorded (2). The ATCC strain was 105

confirmed to be an HD-1 strain of B. thuringiensis by comparison of plasmid profiles to 106

previously published work (41) and by whole-genome sequence analysis with in silico MLST, 107

AFLP and cry gene typing. Unless otherwise indicated, strains were grown on brain heart 108

infusion agar (BHI) containing polymyxin B (50 U/ml) and either spectinomycin (250 μg/ml) or 109

kanamycin (20 μg/ml). Unless otherwise notated, strains were incubated at 30º C. 110

Identification of a Barcode insertion points – Potential insertion sites for the barcodes were 111

identified based on a set of selection rules elaborated in Table 2. Insertion points were identified 112

in the published genome sequence of Btk strains BMB171 (20) and T03a001 (RefSeq accession 113

number BC_CM000751.1). Annotations of the BMB171 genome generated in RAST (5) and 114

PATRIC (16) were compared. We also generated a draft genome sequence of ATCC 33679 (M. 115

Krepps et al., manuscript in preparation) and verified that the genome structure fulfilled the 116

appropriate criteria. Of 294 intergenic regions >500 bp long (Table S1), three potential target 117

insertion points were identified (Table 3) that fulfilled all of the set criteria. 118

Barcode module design – We appropriated a set of published 20 bp tags previously used in 119

signature-tagged mutagenesis studies of pooled yeast strains (33). Tags were individually 120

screened against the Btk genome sequences to eliminate sequences that had homology to any 121

portion of the Btk chromosome. One tag was adopted as a common tag to be shared among 122

multiple strains, while the others were selected as a strain-specific tag (S1, S2, etc.). The tags 123

were flanked by an EcoRI restriction site to facilitate screening of recombinant strains. Figure 1 124

shows the general features of a Barcode module and the design of associated real-time PCR 125

assays. 126

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Barcode insertion – Barcodes flanked by ~750 bp of chromosomal DNA sequence were 127

generated synthetically (DNA2.0, Menlo Park, CA) and cloned into pRP1028 which was 128

delivered by a protocol adapted from the work of Janes and Stibitz (24). The resulting plasmids 129

were delivered by biparental mating into ATCC 33679. Replication of pRP1028 was suppressed 130

by maintaining strains at 37º C. Strains which had integrated the plasmids by homologous 131

recombination were selected on spectinomycin plates. Fluorescence of integrant colonies due to 132

the turbo-rfp encoded on pRP1028 was checked by transillumination. The I-SceI expressing 133

plasmid pSS4333 was delivered by triparental mating into the integrant strains. Green-134

fluorescing, SpcS colonies were screened for the presence of the barcode by PCR amplification 135

and EcoRI digestion of the target locus. pSS4333 was cured by serial transfer on solid media in 136

the absence of selection. The curing of the plasmids was verified by checking the strains for the 137

absence of red or green fluorescence and by the lack of PCR amplification of plasmid-borne spc 138

and kan antibiotic resistance genes. 139

Real-time PCR assays – Primers and concentrations used for the strain construction, verification, 140

and detection of the barcodes are shown in Supplementary Table S2. Barcodes were detected by 141

real-time SYBR® Green PCR assays in 20µL volumes in 384-well optical PCR plates. 142

Amplification, data acquisition, and data analysis were carried out on an Applied Biosystems 143

model 7900HT sequence detection system (Applied Biosystems, Foster City, CA). The barcode 144

reactions were set up using SYBR® Green PCR Master Mix (Cat. 4309155; Applied 145

Biosystems, Foster City, CA), forward and reverse primers, nuclease-free sterile water, and 1µL 146

extracted DNA product. The thermocycler conditions for the Common tag and Barcode 2 were 147

as follows: 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and 60°C 148

for 1 minute, followed by a disassociation stage of 95°C for 15 seconds, 60°C for 15 seconds, 149

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and 95°C for 15 seconds. The Barcode 1 thermocycler conditions were set up similarly to 150

program above, with the following exception: annealing temperature was set to 55ºC for 15 151

seconds (instead of 60ºC for 15 seconds). The linear range for each reaction was determined by 152

developing a standard curve for eight 10-fold serial dilutions of the corresponding genomic 153

DNA. The efficiency of each reaction was calculated from the resulting graphs. 154

Barcode stability – A single starter culture of each strain was grown in 5 ml of media. Three 155

independent 50 ml cultures of each Btk strain (Wild-type, and two strains containing different 156

Specific tags in Target 1) were grown in BHI medium in shaking flasks at 30º C. Each day, at 157

approximately the same time, cultures were diluted 1:1000 into fresh media. The process was 158

repeated for 5 days, after which the cultures were allowed to incubate at 30º C for 3 days in order 159

to induce sporulation. This cycle was repeated each week for 6 weeks, representing 160

approximately 300 doublings. Where applicable, growth was monitored by optical density at 161

600 nm (OD600). 162

Comparative growth of barcoded strains – Growth of the barcoded strains was conducted either 163

in a 20 liter fermenter or in parallel flask culture. For parallel flask cultures, samples were 164

withdrawn periodically for determination of the OD600. For growth in the fermenters, wild-type 165

and barcoded strains were grown in 20 liter volumes of NZ Amine A medium in Micros 30 166

fermenters (New Brunswick Scientific, Enfield CT). Starter cultures of 500 mL were grown in a 167

2 L flask in a shaking incubator until the OD600 reached ~0.4. Seed cultures were aseptically 168

transferred into the Micros 30 liter fermenter (with 20 L working volume) containing the NZ 169

Amine A medium. The operating conditions for the Micros 30 L were controlled with agitation 170

speed of 300 rpm, airflow of one air volume per liquid volume per minute, at 30oC and pH 7.0. 171

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Dissolved oxygen (% of saturation) and optical density (600 nm) were monitored using an in-line 172

probe or by periodic sampling, respectively. 173

Genomic characterization of barcoded strain – A 454 shotgun draft sequence of the barcoded 174

strain was generated by standard methods using the 454 Titanium package (Roche/454, 175

Bramford, CT). Reads were mapped to the scaffolds from the parent strain that had been 176

generated from melded 454 shotgun and paired end libraries using Newbler v2.6 (M. Krepps et 177

al., in preparation). Reads were mapped to the parent strain using the mapping algorithm in 178

Genomics Workbench from CLC Bio (Aarhus, Denmark) using the default parameters. Regions 179

of low or absent sequence coverage were identified and deletion endpoints, where applicable, 180

were identified by manual inspection of the mapped data. 181

Results 182

Integration of Barcodes – We selected Btk strain ATCC 33679, a prototypical HD-1 strain of Btk 183

for our barcoding efforts. Our selection was guided by the long history of the use of HD-1 184

strains as EPA-approved biopesticides, dating as far back as 1961 (23). Btk HD-1 is the active 185

ingredient in Foray®, a commercial Btk product. Target sequences were identified by PCR in 186

both ATCC 33679 and a sample of Foray® (Figure 2A). Barcoded target constructs were 187

synthesized and successfully integrated at two of the three identified loci (Targets 1 and 2). PCR 188

amplification of Target sequences revealed the presence only of EcoRI-digestible product and 189

not the parent product, indicating successful replacement of the parental allele (Figure 2B). 190

Similar results were obtained for Target 2 (data not shown). Repeated attempts to integrate a 191

Barcode into Target 3 were unsuccessful for reasons that are not clear at this time. 192

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Real-time PCR detection assay – To allow facile detection of the barcoded strains, real-time 193

SYBR® Green PCR assays were developed against the Common and Specific tags. The assay 194

directed at the Common tag recognized both barcoded strains, whereas the assays directed at the 195

Specific tags recognized only their cognate strains. Because one of the primers for each 196

sequence is derived from endogenous genetic material, careful control over primer 197

concentrations and PCR amplification conditions was found to be critical to avoid spurious false-198

positive signals due to asymmetric amplification. After careful optimization, none of the assays 199

directed at the barcoded strains recognized the wild-type strain. Figure 3A shows representative 200

real-time PCR assay traces and standard curves (Figure 3B) for each assay. Table 4 lists the 201

linear range and limit of detection of each assay, along with its calculated efficiency. Based on 202

the 11.2 Mbp estimated genome size obtained from the Newbler de novo assembly, which 203

weights the sequence coverage of each element rather than the total size of the assembly, the 204

detection limit for each assay is approximately 8-80 genome copies. The differences in the limit 205

of detection between the assays are most likely attributable to the differences in GC content 206

between the chromosomal primer binding sites. 207

Near-neighbor panel screens/inclusivity and exclusivity – Using the same real-time SYBR® 208

Green PCR assays discussed above, we tested the Barcode PCR assays for specificity against the 209

barcoded strains themselves, their wild-type parent strains, and a selection of related and 210

unrelated bacterial strains (Table 5). The barcode assays were specific for their cognate targets 211

and did not yield amplicons in the unmarked and near-neighbor strains. When non-specific 212

amplification was observed these amplicons produced dramatically higher CT values and no 213

discernible thermal dissociation curves (see Supplementary File S2). 214

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Barcode stability and comparative growth experiments – To evaluate the stability of the 215

integrated Barcodes, tagged strains were grown in pure cultures for 6 weeks of daily passage in 216

50 ml shaking cultures during the week, and were allowed repeatedly to enter sporulation every 5 217

days. This serial transfer experiment was equivalent to approximately 300 bacterial doublings. 218

Large population bottlenecks (~107 cells) at each transfer were chosen to maximize the chance 219

that, had the barcode inadvertently introduced an unfavorable phenotype, a strain containing a 220

compensatory mutation (such as a deletion) would be randomly sampled during the passage 221

experiment. For passaged and input strains, PCR assays directed at the barcodes yielded 222

equivalent CT values given identical quantities of input DNA (data not shown), indicating that 223

the barcode had integrated stably into the chromosome and was not generating selective pressure 224

against its retention. 225

We also compared the in vitro growth of barcoded and parental strains. Figure 4 shows the 226

results of 20 liter fermentations of tagged and parent strains. Both optical density and oxygen-227

consumption growth trajectories were very similar, and sporulation frequencies at the conclusion 228

of both runs were identical and close to 100% (data not shown). Although we did not carry out 229

replicate runs in the fermenter, we performed confirmatory growth curve experiments in the flask 230

cultures during the early phases of the serial passage experiment described above. Like the 231

growth in fermentation culture, the growth curves in flask culture were superimposable and were 232

identical across three parallel cultures for each strain. 233

Genome Resequencing – to identify any other potential genetic alterations to the barcoded strains 234

and to verify unambiguously the location and uniqueness of the barcode insertion, we 235

resequenced one of the barcoded strains and mapped the data a Newbler assembly of the ATCC 236

33679 strain. The barcode insertion points were clearly evident at the specified locus (Figure 237

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5A,B), our re-sequencing data indicated that approximately 350 kb of genetic material had been 238

lost at some point during the strain construction process. Most of the deleted material 239

corresponded to three of the scaffolded regions annotated as plasmids. Bioinformatic analysis of 240

the annotated features generated in RAST and comparison with the deleted genes (Table S3) 241

revealed that most of the deleted material was likely one or more of the many plasmids present in 242

Btk and B. cereus strains (4, 22, 35, 37). Genes lost included many homologues of genes from 243

on BA plasmid pXO1 (35). These plasmids, including pXO1 itself from BA, are readily cured 244

during growth at higher temperatures (3, 39, 43), and given the requirement of prolonged 37ºC 245

incubation to suppress plasmid replication during the homologous recombination phase of strain 246

construction, loss of such material is not surprising. 247

Discussion 248

We have successfully introduced small genetic Barcodes – short, specific identifying signatures - 249

into the genome of Btk and coupled the integration of those signatures to specific real-time PCR 250

detection assays directed toward those barcodes. Our work differs from the widely used 251

“signature tags” that uniquely identify transposon insertions and track abundance of individual 252

mutant pools in large populations (21, 33), in that we aim to tag a single locus in multiple 253

isolates with multiple stable chromosomal tags. Our work is similar in intent to the efforts in the 254

synthetic biology community that added specific “watermarks” to differentiate synthetic 255

genomes from their natural counterparts (14-15), which sought to incorporate simple, neutral 256

signatures into the chromosome as means of uniquely marking a strain. In fact, our efforts 257

expand upon the idea of watermarking strains by developing specific detection assays based on 258

real-time PCR. Indeed, these assays allow the detection and differentiation of barcoded Btk 259

strains both in the laboratory and in the field (Emanuel et al., in preparation). 260

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The ability to assign a specific marker to a strain used in field release studies has additional 261

potential advantages, particularly with viable bioinsecticides. Like simulant releases in heavily 262

used proving ground areas, attempts to attribute infections with Bacillus spp. in areas of 263

widespread Btk application to the Btk serotype actually applied to the treated area have been 264

confounded by ubiquitous environmental Bt isolates that cannot always be unambiguously 265

distinguished from the biopesticide strain ((44) and references therein). The Barcodes endow the 266

new simulant strains with a unique signature that can definitively exclude simulant Btk strains as 267

causative agents of suspected Bt infections that might coincide with simulant releases, and if 268

adopted by commercial pesticide manufacturers, could serve as an exclusionary marker for the 269

biopesticide strains. Furthermore, insertion of the tag into the chromosome minimizes chances 270

of transfer to other strains or species, as the rates of transfer of chromosomal loci between B. 271

thuringiensis strains is quite low (3). 272

We acknowledge that the process used to insert the barcode may have caused the curing of one 273

or more plasmids and/or the loss of chromosomal material. In previous years, the ability to 274

perform post-hoc genomic characterization of mutagenized strains was cost-prohibitive. 275

However, modern whole-genome sequencing allows the detection of such events and allows the 276

precise inventory of genomic content of product strains. The loss of genetic material during in 277

vitro culture of Bacillus strains is not surprising – plasmid are often unstable at high temperature 278

and the strains carry a complement of genetic material that may be unfavorable during in vitro 279

growth in rich media. Recombination of T1B2 into the chromosome during the first step of 280

homologous recombination occurred with much lower frequency than T1B1 (data not shown); in 281

fact, the only successful integrant obtained was the deletion construct presented here. In 282

contrast, multiple successful integrants were obtained for T1B1. Together, these results suggest 283

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that one or more elements of T1B2 may be incompatible with a plasmid-encoded functionality, 284

most likelyputative restriction endonuclease encoded within the 400 kb of deleted material. 285

While our barcode does not contain any obvious candidates for a restriction endonuclease 286

recognition site, we cannot exclude the possibility that it may be sensitive to an endonuclease 287

activity that is specific to a sequence motif present only in Barcode 2; the most likely candidate 288

at this time is the GATC consensus Dam methylation site present in Barcode 2 (Figure 1). While 289

the effect of the loss of ~400 kb of genetic material (Supplementary Table S3) in our strains is 290

not immediately evident, inferences might be gained from studies of plasmid loss in BA. In 291

particular, loss of pXO1 from BA strains is associated with numerous phenotypic changes, 292

including changes to sporulation kinetics, nutritional requirements, and phage sensitivity (42). 293

Our strain also had lost a suite of genes involved in the biosynthesis of zwittermicin, a 294

biologically active compound that, among other activities, potentiates the activity of crystal 295

toxins in insect hosts (7, 27). The loss of this gene cluster containing large polyketide synthase 296

modules is reminiscent of the early loss of surfactin biosynthesis during the domestication of B. 297

subtilis and BG strains (13, 30-31). Based on our phenotypic analysis, the effects of the loss on 298

in vitro growth, colony morphology, and sporulation of the Barcoded strains appear to have been 299

minimal. We are attempting to recreate the barcoded strains to retain and/or restore as much of 300

the full complement of genes as possible. 301

The ability to differentiate two tagged strains based on the site-specific integration of specific 302

genetic tags will allow controlled studies in situations where variables previously could not be 303

controlled. For example, it is anticipated that two strains could be prepared or disseminated 304

using different methods, released and collected simultaneously under identical environmental 305

conditions, then tracked independently in a single set of samples. Our data indicate that 306

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simultaneous detection and quantitation may be possible in mixtures containing wild-type and 307

barcoded strains. Alternatively, a test area could be reused quickly without having to wait for the 308

detection signals to return to background levels. 309

We believe that our barcoding strategy will be generally applicable to genetically tractable 310

microorganisms, although the specific barcode sequences, the genetic tools required to deliver 311

barcodes, and the actual insertion points for the barcodes themselves will differ from organism to 312

organism. These variations will be based on the overall and local genetic structure of the target 313

organism. We are currently automating the bioinformatic identification of barcode insertion 314

points and the design of barcode modules to maximize specificity, sensitivity, and selectivity 315

across a broad range of potential target organisms. 316

Acknowledgements 317

This work was supported by the Defense Threat Reduction Agency (DTRA) project number 318

CB3654 to H.S.G. Sequencing of the Btk isolate was supported by DTRA project number 319

CB2847 to H.S.G., C.N.R, and E.W.S. Thanks to Dr. Jason Edmonds and Dr. Vipin Rastogi for 320

critical reading of the manuscript. The opinions stated in this article are those of the authors and 321

do not represent the official policy of the US Army, Department of Defense, or the Government 322

of the United States. Information in this report is cleared for public release. 323

324

Figure Legends 325

Figure 1 - Barcode module design. A) Barcode modules are integrated into the chromosome 326

between convergently transcribed genes or operons (long filled arrows) in intergenic regions 327

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larger than 500 bp. Primers (arrows) are designed to amplify a ~1500 bp flanking region 328

surrounding the barcode to verify the insertion. B) Barcode modules consist of two 20-329

nucleotide tags flanking an EcoRI restriction site. Real-time PCR assays are designed such that 330

one of the tags serves as a primer binding site (arrows) to generate an amplicon using a second 331

primer that anneals to a region of the chromosome flanking the barcode module. One of the tags 332

(the Common Tag) is present in all barcoded strains, with a second tag (the Specific Tag) that 333

serves as a unique identifier for each individual strain. 334

Figure 2 – Integration of the barcodes. A) Verification of the presence of target regions in two 335

B. thuringiensis HD-1 variants. Target regions were amplified from genomic DNA from the 336

wild-type strain used to make the barcoded constructs (ATCC 33679) and from Foray®, a 337

commercial Btk product. B) PCR verification of barcode insertion. Target region amplicons 338

from each strain and the suicide plasmid (pT1B1) were digested with EcoR1. The presence of 339

the barcode renders the amplicon susceptible to digestion with EcoRI. 340

Figure 3 – Real-time PCR assay for barcode detection. Genomic DNA from each cognate 341

strain was used as the template for real-time PCR assays using barcode-specific primers. Cycle 342

amplification plots (top panels) and standard curves (bottom panels) for each SYBR® Green 343

real-time PCR assay are shown. 344

Figure 4 – In vitro growth of wild-type and barcoded strains in 20 L fermenter. Wild-type 345

(BtkW, open symbols) and barcoded (BtkB, closed symbols) strains were grown in 20 L 346

fermenters as described in Materials and Methods. Optical density (OD) and dissolved O2 (DO) 347

were monitored over the course of the fermentation. 348

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Figure 5 – Whole-genome sequencing to verify barcode insertion. 454 shotgun sequencing 349

reads were mapped onto the draft genome sequences of the wild-type (top) and in silico-modified 350

barcoded genomes (bottom). Perfect matches to reference sequence (top row in each panel) are 351

indicated by dark letters; imperfect matches are indicated by lightly shaded letters. 352

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Table 1 – Strains and Plasmids used in this work 353

Strain Description Source/Reference

Bacillus thuringiensis serovar kurstaki ATCC 33679 HD-1 Biopesticide strain ATCC

T1B1 ATCC 33679 ΔpHD1-XO1; Barcoded at Target 1 with Common tag and Specific Tag 1

This work

T1B2 ATCC 33679 ΔpHD1-XO1; Barcoded at Target 1 with Common tag and Specific Tag 2

This work

Foray® Commercial HD-1 biopesticide product dispersed in Fairfax County, VA

(45)

Escherichia coli E. coli SM10 E. coli donor strain (24)

E. coli SCS110 pSS4333 donor strain (24)

Plasmid Description Reference

pRP1028 Allelic exchange vector, turbo-rfp, SpcR (24)

pSS4332 I-SceI expression vector, gfp, KanR (24)

pT1B1 pRP1028 containing Target 1 with Common tag and Specific Tag 1

DNA 2.0

pT1B2 pRP1028 containing Target 1 with Common tag and Specific Tag 2

DNA 2.0

354

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Table 2 – Selection rules for Barcode Insertion Points 356

Rule Purpose Target region must be located in the chromosome

Maximize stability by incorporation on major replicon

Insertion point must lie near the midpoint of an intergenic space larger than 500 bp

Minimize disruption of potential coding sequences or regulatory elements

No annotated genes or potential ORFs in the intergenic space

Minimize disruption of potential coding sequences or regulatory elements

Must lie between two convergently transcribed genes

Minimize disruption of potential coding sequences or regulatory elements

No repetitive structure in intergenic space Facilitate synthesis and cloning of constructs and minimize potential issues with homologous recombination

No identical repetitive elements >200 bp in size within 10000 bp

Minimize potential loss by deletion via homologous recombination between repeat elements (e.g. insertion sequences)

Target must be intact and consistently annotated in two or more available Btk sequences and in ATCC 33679 draft

Maximize likelihood of success in selected target strain

Target must be present in commercial Btk isolate

Maximize versatility and adaptability of barcode targeting vectors to different strains

357

Table 3: Potential Barcode Insertion Points 358

*PATRIC annotation; no NCBI locus tag called for this gene. 359

360

361

Intergenic Locus (BMB171

coordinates) 5’ flanking gene 3’ flanking gene

Intergenic gap size

(bp)

Target 1 882263-882815 BMB171_C0768 acyl-CoA synthetase

BMB171_C0769, hypothetical protein 552

Target 2 1533619- 1534178

BMB171_C1412, thiosulfate sulfurtransferase

VBIBacthu14800_1517*, hypothetical protein 559

Target 3 2786602- 2787119

BMB171_2615, hypothetical protein

BMB171_C2616, alkane-sulfonate monooxygenase ssuD

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362

Table 4 – Sensitivity of PCR assays for Btk strains 363

Assay Efficiency Linearity Estimated LOD1 (genome copies)

Common Tag 75% 1pg – 10 ng 83 Specific Tag 1 72% 1pg – 10 ng 83 Specific Tag 2 77% 100fg – 10 ng 8.3

1Limit of detection 364

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366

Table 5 - Specificity of real-time PCR assays for Btk strains 367

Assay Common Tag Specific Tag 1 Specific Tag 2

Str

ain

B. thuringiensis serovar kurstaki

0/4 0/4 0/4

B. thuringiensis serovar kurstaki T1B1

4/4 [22.0] 4/4 [25.2] 0/4

B. thuringiensis serovar kurstaki T1B2

4/4 [27.2] 0/4 4/4 [22.0]

Bacillus cereus HER1414 0/4 0/4 0/4 B. anthracis Ames 0/4 0/4 0/4

B. anthracis NNR-Δ1 0/4 0/4 0/4 B. anthracis ΔSterne 0/4 0/4 0/4

B. thuringiensis serovar israelensis ATCC 25646

0/4 0/4 0/4

Bacillus cereus HER1414 0/4 0/4 0/4 B. subtilis ATCC 27370 0/4 0/4 0/4

B. atrophaeus var. globigii (“BG”)

0/4 0/4 0/4

Pseudomonas aeruginosa PAO-1

0/4 0/4 0/4

Streptococcus pyogenes ATCC 12384

0/4 0/4 0/4

Bordetella pertussis ATCC 9797

0/4 3/4 [39.0]* 0/4

Salmonella typhimurium ATCC 14028

0/4 0/4 0/4

Escherichia coli ATCC 43985

0/4 0/4 0/4

Human Placental DNA 0/4 1/4 [39.8]* 0/4 Escherichia coli O157:H7 0/4 0/4 0/4

Francisella tularensis SHU4

0/4 0/4 0/4

Yersinia pestis HARBIN35 0/4 0/4 0/4 Data listed as number of samples crossing threshold per number of replicates tested 368 1 ng of genomic DNA was tested per replicate 369 [# ] represent Average CT values 370 *indicates negative dissociation curve data 371

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